NEW BIOCATALYTIC PLATFORMS FOR THE SYNTHESIS OF HIGHLY VALUABLE COMPOUNDS THROUGH INTENSIFIED BIOPROCESSES
This PhD thesis is focused on developing and optimising biocatalytic platforms to obtain high valuable products in a more sustainable way compared to classical chemical synthesis, focusing the research on alcohols oxidation, synthesis of nitriles and de-glycosylation of glycosides. A particular effort has been devoted towards the study of the best immobilisation solutions for the different biocatalysts employed, in order to maximise the productivity and stability of the platform, using flow reactors in some biotransformations. This thesis has been structured in four different chapters: At first, this thesis focused on the use of a whole-cell system to perform biooxidations of benzyl alcohols mediated by endogenous dehydrogenases of acetic acid bacteria (AAB), getting access to the corresponding carboxylic acids. As key study, I studied the selective de-symmetrisation of 1,4-phenylenedimethanol to the corresponding monoacid 4-hydroxymethyl benzoic acid, an important building block in organic chemistry. The employment of a whole-cell immobilisation approach inside alginate beads allowed an efficient development of a flow system to selectively obtain the desired monocarboxylic acid in a continuous way, avoiding byproducts and the formation of the unwanted dicarboxylic acid (terephthalic acid). Secondly, I focused my attention on the development of an effective synthesis of nitriles avoiding cyanation agents or other toxic compounds by performing a simple dehydration of oximes in water. This process, possible thanks to aldoxime dehydratase enzymes (Oxds), has been optimised to overcome the limited stability of these groups of biocatalysts as well as to maximise productivity. Thanks to a whole-cell entrapment into an alginate matrix, the operational stability of this system was significantly improved. The employment of a flow reactor has further increased the productivity of this biotransformation. Thirdly, I developed a photo-bio cascade to synthesise benzonitriles starting from benzyl alcohols employing a telescopic approach. This project, done during my stay at the University of Oviedo, Spain, under the guidance of Professor Vicente Gotor-Fernández, resulted in the efficient coupling of a photo oxidation of alcohols to aldehydes and the simultaneous condensation of hydroxylamine to obtain oximes in a one-pot reaction. This first step is followed by an enzymatic dehydration of the resulted oximes into the corresponding nitriles, allowing an efficient two-step synthesis of variously substituted benzonitriles. The mild conditions used, as well as the demonstrated possibility to scale up the process, represent significant advantages to prefer this synthetic approach over classical ones. Eventually, I further valorised AAB, not only as biocatalysts but also as producers of bacterial cellulose (BC), a biocompatible and sustainable polymer, which can be turned, by simple chemical functionalisation, into an efficient support for enzymatic immobilisation. As a key study to demonstrate the sustainable nature of this process, I valorised the apple waste as a valuable medium to produce high quantities of BC functionalised to immobilise an extremophilic β-glycosidase to perform a selective de-glycosylation of the apple-derived phloridzin into phloretin, an important bioactive molecule. Particular attention has been drawn on the solvent selection and the process conditions: the use of novel green solvent 2,2,5,5-Tetramethyloxolane (TMO) in a biphasic system allowed a remarkable productivity improvement, nullifying byproducts (usually obtained using classical chemical hydrolysis) and granting an almost “zero-waste” process.
Biocatalysi, Flow chemistry, Flow-biocatalysi, Agro-waste valorisation, enzyme, aldoxyme dehydratase, glycosidase, Photochemistry, Chemo-enzymatic cascade, enzyme immobilisation, whole-cell immobilisation, Settore CHEM-07/C - Chimica e biotecnologia delle fermentazioni, Settore CHEM-05/A - Chimica organica, Settore CHEM-07/B - Chimica degli alimenti, Settore CHEM-04/A - Chimica industriale, Settore BIOS-07/A - Biochimica
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